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Abstract

Background

Results

Examination of nasal and vaginal swabs collected from 12 diseased buffaloes led to
the isolation of three paramyxovirus isolates from two animals. Antigenic, morphological
and biological characteristics of these three isolates were essentially similar to
those of members of the Paramyxoviridae family. Antigenic analysis by direct immunofluorescence and cross neutralization
test placed these isolates together with bovine parainfluenza virus type 3 (BPIV3).
Nucleotide and amino acid phylogenetic analysis of partial matrix gene sequences of
the buffalo isolates and six field BPIV3 isolates from bovines in Argentina were studied.
Buffalo isolates were similar to genotype B (BPIV3b) while the six BPIV3 isolates
were similar to genotypes A (BPIV3a) and C (BPIV3c).

Conclusions

This is the first characterization of BPIV3 in water buffalo.

According to the samples analyzed, in Argentina, the genotype B was found in buffalo
and the genotypes A and C were found in cattle.

Background

Paramyxoviruses are well-known pathogens of the central nervous and respiratory systems
of many host species. In the last few decades, many novel paramyxoviruses have emerged
causing devastating illnesses in different aquatic and terrestrial animals, including
in some cases a species jump to humans [1]. Parainfluenza virus type 3 (PIV3) is an enveloped, single-stranded negative sense
RNA virus within the Respirovirus genus of the Paramyxoviridae family [2,3]. The Respirovirus genus includes human parainfluenza virus types 1 and 3 (HPIV1 and HPIV3, respectively),
Sendai virus (murine PIV1) and bovine parainfluenza virus type 3 (BPIV3) [2]. In some instances when animals are subjected to high stressful conditions, infection
with BPIV3 can contribute to tissue damage and immunosuppression, resulting in severe
bronchopneumonia from secondary bacterial infections [4]. The resulting disease is part of the bovine respiratory disease complex (BRDC) and
is considered as the most significant illness associated with feedlot cattle in the
USA [5], and possibly worldwide. Other respiratory viruses such as bovine herpesvirus 1,
bovine viral diarrhea virus (BVDV) and bovine respiratory syncytial virus (BRSV) have
also been associated with BRDC development in feedlot cattle [6]. The clinical presentation of bovine infections with BPIV3 can vary considerably,
ranging from asymptomatic infections to severe respiratory illness. In most cases
where BPIV3 is implicated in disease, usual clinical signs include coughing, fever
and nasal discharge [6].

Three genotypes, A (BPIV3a), B (BPIV3b) and C (BPIV3c) have been described, based
on genetic and phylogenetic analysis [7]. The BPIV3b genotype could hypothetically be a lineage from a strain that recently
crossed from another host species into cattle [8]. PIV3 infections were found in a wide variety of mammals including cattle, humans,
sheep [9], goats [10], bison [11], guinea pigs [12], black and white rhinoceros [13], moose [14], bighorn sheep [15] and camels [16]. Cross-species infections have been reported in numerous instances, including HPIV-3
in guinea pigs [12], BPIV3 in a human, BPIV3 in sheep, and ovine PIV3 in cattle [17]. In Argentina, serological studies conducted in 1980 and 1984 showed a high incidence
of antibodies against BPIV3 (77%) in cattle from the main livestock breeding regions
[18,19]. In addition, positive serology was reported in domestic and wild South American
camelids [20-22] and the virus was isolated from cattle and sheep [23]. However, little is known about the circulation of BPIV3 in cattle and other ruminants
in Argentina, as well as about the genotypes that are present in this Southern American
country.

Production systems have evolved to mixed managements, where alternative production
species, such as the water buffalo (Bubalus bubalis), coexist in the same habitat as with cattle. Water buffalo breeding represents an
important economic alternative in Argentina, which allows access to national and international
markets. This species is susceptible to several viral infections [20,24,25] including BPIV3 as reported in a very early in 1966 in Egypt [26]. In addition to its potential relevance with respect to water buffalo health, this
finding bears epidemiological significance due to the risk of transmission of the
virus to cattle. The aim of this study was to characterize antigenically and genetically
BPIV3 isolates from a respiratory and reproductive syndrome outbreak in dairy buffaloes
and compare the buffalo isolate with contemporaneous BPIV3 circulating in cattle.

Results

Morphological and biological properties of buffalo viral isolates

PIV3 was successfully isolated from two out of twelve water buffaloes studied, in
one case from both nasal (7 N) and vaginal (7 V) swabs, and in the other only from
the vaginal swab (2 V).

Isolates produced characteristic cytopathic effects (CPE) on MDBK cells, similar to
those of the BPIV3 reference strain, with many scattered, rounded, refractory cells
and small syncytia. CPE were first visualized on the second passage, after three days
post inoculation (DPI), and CPE were observed at day 4 or 5 DPI. Both bovine polyclonal
anti-BPIV3 serum and FITC-anti-BPIV3 IgG reacted with the cells infected by these
isolates (Figure 1 A).

Transmission electron microscopy revealed spherical to pleomorphic virions, approximately
100–300 nm in diameter, which were morphologically indistinguishable from paramyxoviruses.
Intact virions were enveloped and densely packed, as has been described for BPIV3.
Nucleocapsids were visible and exhibited a typical “herringbone” pattern (Figure 1 B).

The three isolates (7 N, 7 V and 2 V) were able to agglutinate red blood cells (RBCs),
with UHA titers of 16, 8 and 4 respectively. The agglutinated RBCs eluted after 1 h
suggesting neuraminidase function/activity. The HI test showed 58% of positive reactions,
with titers ranging between 1.6 and 2.5 (data not shown).

Molecular characterization RT-PCR and sequencing

A fragment of the M gene, consistent with the expected size of 328 bp was amplified
from the 3 virus isolates (7 N, 7 V and 2 V) by RT-PCR. As expected, the primer pairs
did not amplify using genomic material of BRSV as template. Analysis of the alignments
of 271 pb of the corresponding sequences revealed that they are related to BPIV3 strain
Q5592 from Australia with 94% nucleotide identity (Figure 2) and 100% predicted amino acid identity (Table 1).

Table 1.Comparison of identity percentages in amino acid and nucleotide sequences of the 217-bp
region of the M gene among buffalo and bovine isolates and reference strains of BPIV3

Data and phylogenetic analysis

Phylogenetic analysis of the M gene fragment of the three Argentinean buffalo isolates
(7 N, 7 V and 2 V) together with other paramyxoviruses, placed the buffalo isolates in the same clade as BPIV3 (Figure 3).

Figure 3.Phylogenetic analysis based on M gene fragment. Phylogenetic analysis was performed using MEGA 4 software with 1,000 bootstrap replicates.
Phylogenetic analysis based on M gene fragment nucleotide sequences compared to other
Respirovirus genus members. The Argentinean isolates are indicated in bold. GenBank accession
numbers are presented in Materials and Methods.

In particular, the isolates grouped with the BPIV3b genotype represented in the tree
by an Australian isolate (Q5592). BPIV3b is distinct from the previously characterized
members of BPIV3 represented by genotypes A and C.

In order to determine whether the PIV3 genotype found in buffalo is also circulating
in cattle, the same gene fragment was amplified by RT-PCR using as template BPIV3
RNA isolated from six bovines of the Buenos Aires region. Three of the six isolates
show 98% nucleotide identity and 100% amino acid identity with BPIV3c (SD0835 strain)
reference isolate, and the remaining three, 99% nucleotide identity and 100% amino
acid identity with BPIV3a (SF strain) reference isolate (Table 1). Phylogenetic characterization showed that the bovine isolates belong to the BPIV3a
and BPIV3c genotypes, which have been reported so far in North America, Europe and
China (Figure 3).

Serum neutralization test

Buffalo sera (n = 24) were analyzed by cross-neutralization, Three were negative for
both viruses, BPIV3a and b, 9 showed higher titers against BPIV3b isolated from buffalo,
4 showed higher titers against BPIV3a and the remaining 11 had same titers for both
viruses. (Table 2). In general, sera from buffaloes showed higher antibody titers against the isolated
buffalo virus than against the reference virus.

Table 2.Comparison of neutralizing titers against BPIV3b isolated from buffaloes and against
a BPIV3a reference strain

Discussion

In the present study, we describe the isolation of BPIV3 from two water buffaloes
with respiratory and reproductive signs. The virus was isolated from nasal and vaginal
swabs from two out of twelve animals with reproductive and respiratory signs studied.
To our knowledge little is known about the pathogenesis of this virus in buffalo.
Should there be a syndrome similar to that present in cattle where the virus is transiently
excreted [27], the unsuccessful virus isolation from the then remaining symptomatic animals could
be related to the time point of sampling, which could have taken place at the end
of the acute infection.

The IF assay showed strong and specific binding of anti-BPIV3 antibodies to cells
infected with these three positive clinical isolates. Sera from buffaloes were able
to neutralize the activity of the 7 N, 7 V and 2 V isolates and that of the reference
bovine virus (BPIV3 strain SF), but the neutralization titers against the latter were
lower than against buffalo isolates, suggesting antigenic differences.

The sequence of the M amplified fragment showed 94% nucleotide identity between buffalo
isolates and BPIV3b strain Q5592 from Australia [8]. This is the first report of molecular characterization of a BPIV3 isolated from
buffaloes. In order to determine homology between PIV3 isolates from Argentinean bovine
and buffaloes, we characterized retrospectively six isolates collected from bovine
respiratory outbreaks that occurred in 2009 and 2011. These isolates showed high percentages
of nucleotide and amino acid identity with A and C genotypes (Table 1). There are only few reports about BPIV3 circulation in Argentina, Epstein et al described the isolation, virological and physicochemical characterization of PIV3
from Argentinean cattle and sheep in 1974 [23]. However, phylogenetic studies of these isolates were not carried out. Phylogenetic
reconstructions based on the alignment of the M-gene nucleotide sequences demonstrated
that the 7 N, 7 V and 2 V isolates clustered in the B genotype group. So far, this
genotype has only been previously reported in Australia [8]. The phylogenetic position of the Argentinean buffalo isolates might be correlated
with the antigenic differences observed by cross-neutralization studies, where the
reference strain belonging to the BPIV3a group was lesser neutralized than the buffalo
viral isolates by sera of buffaloes. Therefore the serological and molecular characterization
of buffalo BPVI3 isolates show that these viruses are more similar to B than to A
(SF reference strain) and C BPVI3 genotypes (SD0835 reference strain). Interestingly,
the genotype B isolated from buffaloes was not found among the bovine samples, analyzed
in this work, that clustered with genotypes A and C. Genotype B was until now observed
only in Australia with the hypothesis of a recent crossing from another species into
cattle [8]. This evidence of genotype B in buffaloes should need further investigation to know
if this species could be one of the host species of this genotype. Therefore analysis
of a larger number of isolates from cattle and buffaloes is needed to determine whether
the three genotypes circulate in both species, or if the distribution observed is
representative. Importantly, this is the first report of bovine B and C genotype circulation
out of Australia and China, respectively. The circulation of genotype C only in China
was attributed to a geographic restriction [7]. This hypothesis can be ruled out by our results and this observation was most likely
due to the relative frequencies of these genotypes with respect to the samples analyzed.

To our knowledge this is the first report of the isolation of a parainfluenza virus
type 3 from vaginal secretions. However, it is important to highlight that the presence
of the virus in the secretions could be due to oro-genital contact between animals.
Evidence of viral replication in the genital mucosa needs new investigations.

Conclusion

The isolation and characterization of BPIV3 from water buffaloes were reported. We
identified the isolates as bovine parainfluenza virus type 3 genotype B that differs
from genotypes A and C circulating in Argentinean cattle herds reported so far. Further
studies would be required to determine if A and C genotypes also circulate in buffalo
herds, as well as to elucidate the role of buffalo as reservoirs, especially of the
B genotype, and in the transmission of this virus to cattle.

Methods

Samples

Nasal and vaginal swabs were obtained from 12 water buffaloes of a dairy farm in the
Argentinean province of Chaco, during an outbreak of respiratory and reproductive
disease in July 2009. These animals had white stringy mucus in the nose and vulva.
These samples were inoculated on Madin Darby bovine kidney (MDBK) cell cultures. The
SF strain of BPIV3 was used as reference. Cells were daily examined under optical
microscope to observe the appearance of cytopathic effects.

Bovine isolates were obtained from cattle with respiratory syndromes, 5 of them in
the year 2009 and the remaining isolate in 2011. They were collected and amplified
from Azul laboratories (Azul-Buenos Aires province) from outbreaks of Buenos Aires
province.

Immunofluorescence test

Virus isolates were inoculated into confluent MDBK cells in 8-well chambers at a MOI
of 1.0, 0.1, and 0.01. For direct IF test, Cells with cytopathic effects were fixed
with 99.5% acetone and then incubated with FITC anti-BPVI3 IgG (Ames). For indirect
IF, cells were incubated with antibovine PIV3 polyclonal antibody (NVSL) diluted 1:100
in PBS at 37 °C in a moist environment for 1 h. Then, cells were incubated with FITC
antibovine IgG (KPL). In both cases, slides were layered with buffered glycerin and
observed under epifluorescence in an Olympus BX 40 + H hal microscope.

Serial two-fold dilutions of culture supernatants from infected MDBK (50 ul) were
titrated against 50 ul Guinea pig erythrocytes in V-bottom microtiter plates (Nunc).
After incubation at 4°C for 4 h, hemagglutination was evaluated by the appearance
or absence of a red cell button. Results were expressed as hemagglutinating units/50 μl
(HAU/50 μl). The last dilution at which hemagglutination was observed was taken as
the endpoint of the hemagglutinating activity. The reciprocal of this dilution expresses
the number of hemagglutinating units or virus titer in UHA.

For the HI assay serial two-fold dilutions of sera from buffaloes were allowed to
react with a fixed dose of reference viral haemagglutinin (8 UHA), followed by the
addition of guinea pig agglutinable erythrocytes. Titers were expressed as the log10
of the reciprocal of the highest dilution hemagglutination inhibitory multiplied by
a constant factor of 8.

RT-PCR and sequencing

Infected MDBK cells were scraped off the plates and homogenized by three cycles of
freezing and thawing. After an initial centrifugation at 3,000 g for 15 min, polyethylene
glycol 8000 (Sigma), 10% (w/v, final concentration) was added to the cell lysates,
followed by incubation for 4 h at 4°C. The virus was pelleted at 12,000 g for 60 min
at 4°C. Viral genomic RNA was extracted from the virus pellet using RNeasy Mini Kit
(QIAgen). Oligonucleotide primers for BPIV3 detection and identification (Mfwd: 5´AGTGATCTAGATGATGATCCA
3´ nt - 3960 and Mrev: 5´GTTATTGATCCAATTGCTGT −3´ nt - 4288) were designed based on
a 328 bp segment of the consensus BPIV3 Matrix (M) gene. cDNA was synthesized using
the specific oligonucleotide primer (Mfw) and MMLV reverse transcriptase (Promega).
Subsequently, Go Taq DNA Polymerase (Promega) was used to amplify the M gene fragment.

Serum neutralization test

Cross-neutralization tests were performed in MDBK 96-well plates, by overnight incubation
of 200 TCID50 on 50 ul of 7 N buffalo virus isolates or BPIV3 reference strain (SF) with 50 ul
heat-inactivated two-fold diluted sera. The plate was incubated for 5 days at 37 °C,
under 5% CO2, and then screened for the presence or absence of cytopathic effects
under optical microscope (400x magnification) to determine the virus-neutralization
titers. Neutralization titers were calculated as the decimal logarithm of the reciprocal
of the last serum dilution with clear cytopathic effects.

Authors’ contributions

SM and SR designed the experiments, analyzed the data and drafted the manuscript together.
SM performed the experiments. PL, GC, JK, and GC gently surrendered the field isolates.
DR and VP kindly provided the reference strain. AM and ET participated in the molecular
genetic studies, interpretation of data and contributed to the manuscript. MC helped
to draft the manuscript. JD helped with electron microscopy. OZ helped with cell cultures.
All authors read and approved the final manuscript.

Acknowledgements

The authors wish to thank Mónica Florin-Christensen (INTA) for helping to draft the
manuscript and Domnique Ziant for their excellent technical assistance (University
of Liege, Belgium). This work was supported by Fonds de la Recherche Scientifique
(FRS-FNRS) and Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET).
SM is a CONICET Fellow (Argentina).